Recombinant vector and use thereof for exocellular preparation of single molecula antibodies from bacillus subtilis

ABSTRACT

A recombinant vector for the expression and secretion of antibodies in single molecule form (scFv) in  B.subtilis , where said vector comprises the promoter of the gene for neutral protease, a new secretion sequence (I) and a DNA sequence coding for an antibody scFv of interest, a strain of  B.subtilis  transformed with said recombinant vector, and a procedure for the exocellular production of scFv antibodies by means of a culture of said strain of  B.subtilis  are described. The recombinant vector allows the expression of scFv in a completely soluble form and its secretion in high yields.

[0001] This invention relates in general to the exocellular productionof antibodies in single molecule form (scFv) from Bacillus subtilis(B.subtilis).

[0002] In particular, this invention relates to a recombinant vectorwhich comprises the promoter of the gene for neutral protease, a newsecretion sequence and a DNA sequence coding for an antibody scFv ofinterest, a strain of B.subtilis transformed with said recombinantvector and a procedure for the exocellular production of scEv antibodiesby means of a culture of said strain of B.subtilis.

[0003] The possibility of obtaining antibody fragments (mini-antibodies)by means of their expression in bacterial cells has opened up new andinteresting application prospects.

[0004] In fact, their reduced dimensions allow an improvement inpharmacokinetic characteristics for diagnostic and therapeutic uses. Inaddition, the production of mini-antibodies in microorganisms iseconomically more favourable than the production of monoclonalantibodies (mAbs) in mammalian cells since greater reproducibility ofthe preparations and a complete absence of possible contaminants fromoncogenic viral DNA are guaranteed. The antibodies are tetramericproteins formed from two heavy chains (H) and two light chains (K orlambda). In the H chain a variable region (VH) and three constantregions may be observed, while in the light chain only one constantregion is present in addition to the variable region (VK). The variableparts, which are at the amino-terminal end of each chain, areresponsible for binding to the antigen whereas the constant portionsinteract with the other components of the immune system (effectorfunction). The regions responsible for binding to the antigen and thosewith an effector function may be separated by enzymatic digestionwithout altering their functionality. Indeed, it is known that the Fvportion of the antibody, ie VH+VK, isolated by means of enzymaticdigestion of IgG, maintains totally its ability to bind to the antigen.

[0005] The use of said portion of the antibody, which forms the entireantigen-binding site, may present some stability problems for the Fv inthat the two chains, not being covalently bound, tend to dissociate.

[0006] Recently some strategies have been proposed to reduce thedrawback of the two chains dissociating.

[0007] Of particular interest appears to be that described by Bird etal., 1988 (Science, 242: 423-426) which consists of the expression inE.coli of a molecule formed by the union of the variable regions of thetwo chains by way of a suitable peptide (linker). The two regions VH andVK are thus synthesized into a single molecule (scFv), with the carboxylend of the VK region bound to the amino-terminal end of the VH, orvice-versa, by means of the linker.

[0008] When using this strategy, however, Bird et al. obtained anexpression of scFv inside the cell in the form of non-functionalinsoluble aggregates. The functionality of these molecules may berecovered only by following complex procedures which, though efficient(Bird et al. report renaturing yields of 5% to 30%), are of no advantagewhen compared with systems where antibodies are produced in a solubleand functional form.

[0009] The known art describes systems for the secretion of functionalscFv in the periplasm of E coli, an environment from which antibodymolecules may be recovered following controlled lysis of the bacteria.

[0010] Currently particular attention is being given to the developmentof systems for the synthesis and secretion of recombinant molecules inB.subtilis.

[0011]B.subtilis is in fact an interesting microorganism from thebiotechnological viewpoint: It is completely non-pathogenic, has theability to secrete the product of gene expression into culture mediumand is easy to cultivate on a large scale.

[0012] A partial limitation on the use of this microorganism is the lackof vectors which allow an efficient exocellular production of scFvantibodies. Bird et al. (U.S. Pat. No. 4,946,778) obtained 1 mg/liter ofscfv using systems of expression and secretion in B.subtilis comprisingthe promoter regions and secretion sequence isolated from the genescoding for proteolytic enzymes.

[0013] Recently a new system for improving secretion of scFv inB.subtilis (Wu et al., 1993, Biotechnology, 11: 71-76) was proposed. Thevalues obtained (5 mg/liter), however, still appeared to be of littleinterest for industrial use.

[0014] It has been found that the inconveniences of the above mentionedtechnology may now be overcome by adopting a particular recombinantvector which comprises the promoter of the gene for neutral protease anda DNA sequence coding for a new secretion peptide.

[0015] In particular, said recombinant vector allows the expression inB.subtilis of scFv antibodies in a completely soluble form and theirsecretion in high yields.

[0016] An object of the present invention is a recombinant vector forthe expression and secretion of antibodies in single molecule form inB.subtilis which comprises the promoter of the gene for neutralprotease, a new secretion sequence and a DNA sequence coding for anantibody in single molecule form.

[0017] A further object of the present invention is a strain ofB.subtilis transformed with said recombinant vector.

[0018] A further object of the present invention is a procedure for theexocellular preparation of said antibodies which comprises thecultivation of a strain of B.subtilis transformed with said recombinantvector and the removal from the culture environment of antibodies insingle molecule form.

[0019] A further object of the present invention is the use of saidantibodies in diagnostics and therapy.

[0020] A further object of the present invention is the DNA sequencecoding for a new secretion peptide signal.

[0021] A further object of the present invention is the antibody scFv5E8specific for sub-unit alpha of human chorionic gonadotropin. Furtherobjects of the present invention will be apparent from the ensuing textand examples.

DESCRIPTION OF THE FIGURES

[0022]FIG. 1: this shows the nucleotide and amino acid sequence of thevariable region of the heavy chain (VH) of the monoclonal antibody 5E8specific for sub-unit alpha of human gonadotropin.

[0023]FIG. 2: this shows the nucleotide and amino acid sequence of thevariable region of the light chain (VK) of the monoclonal antibody 5E8specific for sub-unit alpha of human gonadotropin.

[0024]FIG. 3: this shows the restriction map of the recombinant vectorpSM507.

DETAILED DESCRIPTION OF THE INVENTION

[0025] In particular, the recombinant vector of the present inventioncomprises:

[0026] 1) the promoter of the gene for neutral protease of B.subtilisBGSC 1A341;

[0027] 2) the secretion sequence (I)

[0028] 5′ ATG AGA AGC AAA AAA ACG CGT ATC AGC TTG TTG

[0029] TTT GCG TTA ACG TTA ATC TTT ACG ATG GCA TTC AGC

[0030] GGC CGC TCT GCC ATG GCC 3′ and

[0031] 3) a DNA sequence coding for an antibody in single molecule formwith the sequence VH/VK-L-VK/VH-(TAG)_(n)

[0032] where:

[0033] VH and VK are the variable regions of the heavy and light chainsof an antibody of interest; L (Linker) is the peptide linker between thetwo variable regions Val-Ser-Ser-(Gly₄-Ser)₃; TAG is a peptiderecognised by polyclonal antibodies directed towards the same peptide, nis 1 or 0.

[0034] The secretion sequence (I), or sequence leader (LS), codes for anew secretion peptide with the following amino acid sequence:

[0035] Met Arg Ser Lys Lys Thr Arg Ile Ser Leu Leu Phe

[0036] Ala Leu Thr Leu Ile Phe Thr Met Ala Phe Ser Gly

[0037] Arg Ser Ala Met Ala.

[0038] The recombinant vector of the present invention may be obtainedby:

[0039] a) synthesis of an oligonucleotide which comprises the secretionsequence (I), the sequence coding for the peptide linker and ultimatelythe sequence coding for the peptide TAG and where said oligonucleotidecontains unique restriction sites which allow the insertion of thesequence coding for the variable region of the heavy or light chain ofthe antibody downstream of secretion sequence (I) and upstream of thepeptide linker and the insertion of the sequence coding for the variableregion of the light or heavy chain of the antibody downstream of thelinkage peptide and upstream of the peptide TAG;

[0040] b) cloning of said oligonucleotide in a plasmid vector comprisingthe promoter of the gene for neutral protease of B.subtilis BGSC 1A341;

[0041] c) cloning of the DNA sequences coding for the variable region ofthe heavy and light chains of the antibody of interest in theoligonucleotide restriction sites; and finally

[0042] d) isolation of the recombinant vector.

[0043] According to one embodiment of the present invention theQligonucleotide in point a) has the sequence (L1):

[0044] EcoRI   5′GAA TTC TTA TGA GAA GCA AAA AAA CGC GTA  30 TCA GCT TGTTGT TTG CGT TAA CGT TAA TCT TCA  63 CGA TGG CAT TCA GCG GCC GCT CTG CCATGG CCG  96                PstI          BstEII CAC AGG TCC AAC TGC AGCCTA TGG TCA CCG TCT 129 CCT CAG GTG GCG GTG GCT CTG GCG GTG GTG GGT 162                                      KpnI CGG GTG GCG GCG GAT CTG ACATTC AAG GTA CCC 195      Bg1II CCT GAG ATC TCA TGG AAG AAC TTA TGA TCGAGG 228            SalI      HindIII GTA GGT AAG TCG ACA AGC TT 3′ 248      Stop

[0045] where: immediately downstream from the secretion sequence (I)(from nucleotides 9 to 95 inclusive) there are 4 restriction sites(PstI, BstEII , KpnI and BglII) positioned in such a way as to be ableto clone in the same reading order the DNA sequences coding for thevariable region of the heavy chain (VH) and the light chain (VK)respectively. Between the BstEII site (C-terminal region of the V_(H))and the KpnI site (N-terminal region of V_(K)), the sequence codes for apeptide linker between the two regions, having the sequenceVal-Ser-Ser-(Gly₄-Ser)₃; furthermore, the sequence which follows theBglII site codes for a nonapeptide with the sequenceMet-Glu-Glu-Leu-Met-Ile-Glu-Gly-Arg useful as a specific target ofrecognition by anti-peptide antibodies for monitoring the expression ofthe protein, using the Western blot method, and for its purification.This sequence is followed by a stop codon for translation and therestriction sites SalI and HindIII.

[0046] In the preferred embodiment, the plasmid vector in point b) ispSM308 ATCC 68047 comprising the origin of replication in B.subtilis,the gene CAT coding for chloramphenicol resistance, the promoter forneutral protease (npr) of the strain B.subtilis BGCS 1A341, an EcoRIrestriction site located immediately downstream of the promoter and theribosomal recognition site (RBS) and a HindIII site upstream from theEcoRI site.

[0047] The cloning of the oligonucleotide may be conducted according tonormal techniques, using a plasmid vector previously digested withsuitable restriction enzymes. Subsequently, from positive clonesobtained by techniques of transformation and selection, cloning plasmidsuseful for the construction of the recombinant vector mav be isolated.

[0048] By operating in preferred conditions, a plasmid vector of about 4Kb is obtained, denominated PSMA, in which L1 is correctly inserteddownstream from the promoter npr.

[0049] The introduction of the sequences coding for the variable regionsof the heavy and light chains of an antibody in the restriction sites ofthe oligonucleotide L1 results in the construction of the recombinantvector which, according to the present invention, starting from a singlepromoter, allows the simultaneous expression in the same cell of acompletely soluble precursor having the following sequence:

[0050] LS-VH-L-VK-TAG

[0051] where:

[0052] LS is the DNA sequence (I) coding for the new secretion peptidesignal;

[0053] VH is the variable region of the heavy chain;

[0054] VK is the variable region of the light chain;

[0055] L is the linkage peptide Val-Ser-Ser-(Gly₄-Ser)₃ and

[0056] TAG is the nonapeptide Met-Glu-Glu-Leu-Met-Ile-Glu-Gly-Arg.

[0057] Said precursor is then processed correctly at membrane level andthe antibody VH-L-VK-TAG is secreted in the culture medium at a highyield.

[0058] The sequences coding for the variable regions VH and VK may beselected from those of monoclonal antibodies specific to an antigen ofinterest.

[0059] By way of non-limiting example of the invention, the codingsequences (genes) for the variable regions of the heavy and light chainsof the monoclonal antibody 5E8 specific for sub-unit alpha of humanchorionic gonadotropin were cloned (A. Albertini et al., 1987 (“HumanTumorMarkers , Ed. Walter de Gruyter & Co., Berlin) 387-401).

[0060] From a hybridoma culture producing monoclonal antibodies 5E8, allmRNA was extracted from which was subsequently amplified the DNA codingfor the variable regions of the light (VK5E8) and heavy chains (VH5E8)of the antibody in question. The operations were conducted following theusual methods employing two pairs of oligonucleotides as primers, whichhybridize at the 5′ and 3′ ends respectively of the genes coding forsaid regions.

[0061] By means of the amplification technique DNA fragments wereobtained which comprised the gene VH5E8 delimited at the 5′ end by aPstI site and at the 3′ end by a BstEII site and the gene VK5E8delimited at the 5′ end by a KpnI site and at the 3′ by a BglII site.

[0062] The DNA fragments digested with the pair of restriction enzymesPstI and BstEII, and KpnI and BglII were cloned in the vector PSMAobtaining the recombinant vector psM507.

[0063] In accordance with the present invention, recombinant vectorscharacterised by combinations of promoters/secretion sequences differentfrom npr/(LSI) were constructed.

[0064] For this purpose the plasmid pSM268 was used, comprising aconstitutive promoter (Pc), the origin of replication in B.subtilis, thegene coding for resistance to chloramphenicol and the EcORI restrictionsites, situated immediately downstream of the attachment enablingsequence for the ribosomes (RBS) for B.subtilis, and HindIII locateddownstream of the EcoRI site. Said plasmid was obtained from pSM214 ATCC67320 by deletion of the region AvaII-XbaI (which comprises the originof replication in E.coli and the sequence Kmr) and subsequentcircularisation with T4 DNA ligase.

[0065] An oligonucleotide (L2) was then synthesized, comprising thesequence of 22 triplets coding for the leader peptide of the gene pelBof Erwinia carotovora (Lei, S. P. et al., 1987, J. Bacteriol. 169, 4379)and having the following sequence:

[0066] EcoRI  5′ GAA TTC ATA TGA AAT ACC TAT TGC CTA CGG  30 CCG CCG CTGGAT TGT TAT TAC TCG CTG CCC AAC  63                               PstICAG CCA TGG CCG CAC AGG TCC AAC TGC AGC CTA  96  BstEII TGG TCA CCG TCTCCT CAG GTG GCG GTG GCT CTG 129 GCG GTG GTG GGT CGG GTG GCG GCG GAT CTGACA 162          KpnI        Bg1II TTC AAG GTA CCC CCT GAG ATC TCA TGGAAG AAC 195                             SalI   HindIII TTA TGA TCG AGGGTA GGT AAG TCG ACA AGC TT 3′ 227                       Stop

[0067] Finally, the construction of the following cloning plasmids wasundertaken:

[0068] PSMB comprising the npr promoter and the oligonucleotide L2(secretion sequence signal of pelB);

[0069] PSMC comprising the constitutive promoter Pc and theoligonucleotide L1 (secretion sequence signal I)-and

[0070] PSMD comprising the constitutive promoter Pc and theoligonucleotide L2 (secretion sequence signal of peiB).

[0071] The cloning in said plasmids of DNA fragments comprising the geneVH5E8 and the gene VK5E8 has led to the isolation of the recombinantvectors pSM438 (npr/pelB), pSM443 (Pc/sequence (I)) and pSM442(Pc/pelB).

[0072] Subsequently, cells of B.subtilis transformed with said vectorswere cultivated in a suitable culture medium. From analysis of theintracellular and exocellular proteins of said culture it was shownthat:

[0073] the plasmid pSM507 gives rise to the accumulation of at least 30mg/liter of soluble antibody in the supernatant;

[0074] the plasmid psM442 is responsible for the expression of highlevels of antibody (10% of total proteins) but almost exclusively ofintracellular and insoluble form;

[0075] the plasmid psM438 results in the secretion of scFv at a quantitythree times less than plasmid pSM507 and gives rise to the accumulationof precursor in an insoluble form, and finally

[0076] the plasmid psM443 does not give rise to accumulation ofprecursor but to a level of secretion lower than that found with plasmidpsM507.

[0077] These results indicate that the combination of promoter of thegene for neutral protease/secretion sequence signal (I) is fundamentalfor optimum expression and secretion of the antibody. Therefore, therecombinant vector according to the present invention is useful for theexocellular production of scFv antibodies by means of a fermentationprocedure which uses a strain of B.subtilis transformed with saidvector.

[0078] Typically the procedure may be carried out by cultivating astrain of B.subtilis transformed with the recombinant vector of thepresent invention in a culture medium containing a source of carbon, asource of nitrogen and microelements, then isolating the antibody scFvsecreted therein.

[0079] Said antibodies may be purified out using one of the techniquesnormally used in this particular field of work.

[0080] Binding and affinity tests carried out on the unpurified cellularextract obtained from the culture medium of B.subtilis cells (pSM507)indicate that interaction characteristics at the binding site of scFv5E8compared with the monoclonal antibody of origin are maintained.

[0081] Therefore the antibody in single molecule form, a further objectof the present invention, may be used in diagnostics for thedetermination of endocrine tumours and, in general, tumours oftrophoblastic origin.

[0082] The plasmid pSM507 was filed as B.subtilis SMS300 (PSM507) at theAmerican Type Culture Collection where it received the filing numberATCC 69173.

[0083] The following examples have the purpose of illustrating thepresent invention without limiting its scope.

EXAMPLE 1

[0084] Construction of Cloning Vector PSMA.

[0085] The plasmid pSM308 ATCC 68047 (10 μg) was digested with theenzymes EcoRI and HindIII (Boehringer) at 37° C. for 1 hour. Thereaction was immediately blocked with EDTA 20 mM (final concentration).

[0086] An aliquot of the digestion mixture was ligated with theoligonucleotide L1, synthesized by means of the System Plus (Beckman)automatic synthesizer, in a ligase mixture (1 mM ATP, 20 mM Tris-HCl pH7.6, 10 mM MgCl₂ and 10 mM DTT) containing 2 U of T4 DNA ligase. Thereaction was conducted at 14° C. for one night. Finally the ligasemixture was used to transform cells of B.subtilis SMS330 (rec+, npr−,apr−) rendered competent according to the method described by Contenteand Dubnau (Mol. Gen. Genet. 167, 251-258, 1979).

[0087] The recombinant clones were selected onto plates of TBAB (DIFCO)containing 5 μ/ml of chloramphenicol (Cm). From a positive clone(Cm^(R)) the plasmid vector named PSMA was isolated.

EXAMPLE 2

[0088] Construction of the Cloning Vectors PSMB, PSMC and PSMD.

[0089] The plasmids pSM308 ATCC 68047 and pSM268 (10 μg) were digestedwith the restriction enzymes EcoRI and HindIII and then ligatedseparately with the oligonucleotide L1 or the oligonucleotide L2. Theligase mixtures were then used to transform suitable cells of B.subtilisSMS330. Finally, from the positive clones selected onto mediumcontaining Cm, the following cloning vectors were isolated:

[0090] pSMB comprising the promoter npr and the oligonucleotide L2(secretion sequence signal of pelB)

[0091] pSMC comprising the constitutive promoter Pc and theoligonucleotide L1 (secretion sequence signal (I)) and

[0092] pSMD comprising the constitutive promoter Pc and theoligonucleotide L2 (secretion sequence signal of pelB).

EXAMPLE 3

[0093] Cloning of the Variable Regions of the Heavy and Light Chains ofMAb5E8 Specific For Sub-unit Alpha of Human Gonadotropin.

[0094] The following primers were utilised for the cloning:

[0095] 1) VH₁FOR having the sequence:

[0096] BstEII

[0097] 5′ TGAGGAGACG GTGACCCTGG TCCCTTGGCC CCAG 3′

[0098] used for annealing to the 3′ terminal of the helix of thesequence coding for the variable region of the heavy chain;

[0099] 2) VK₁FOR having the sequence:

[0100] BglII

[0101] 5′ GTTAGATCTC CAGCTTGGTC CC 3′

[0102] used for annealing to the 3′ terminal of the helix of thesequence coding for the variable region of the light chain;

[0103] 3) VH1BACK having the sequence:

[0104] PstI

[0105] 5′ AGGTIIAICT GCAG(G/C)AGTCI GG 3′

[0106] used for annealing to the 3′ terminal of the counter-clockwisehelix of the sequence coding for the variable region of the heavy chain;

[0107] 4) VKBACK having the sequence:

[0108] KpnI

[0109] 5′ GACATTCAGG GTACCCAGTC TCCA 3′

[0110] used for annealing to the 3′ terminal of the counter-clockwisehelix of the sequence coding for the variable region of the light chain.

[0111] A) Preparation of cDNA

[0112] The hybridoma which produces the monoclonal antibodies 5E8specific for sub-unit alpha of human gonadotropin was cultivated in themedium RPMI 1640 supplemented with foetal bovine serum at 10%,L-glutamine 200 mM, penicillin and streptomycin and about 4×10⁷ cellswere used to isolate all the RNA. The mRNA polyA+ was separated from theremainder by an extraction technique using guanidine-isothiocyanate (RNAextraction kit supplied by Stratagene) followed by affinitychromatography on oligodT cellulose (Boehringer).

[0113] With the object of cloning the sequence coding for the variableregion of the heavy chain, 25 μl of a reaction solution containing 5 μgof mRNA, 20 pmoles of the primer VH₁FOR, 250 μM of each of DATP, dTrP,dCTP and dGTP, 10 mM of dithiotreitol (DTT), 100 mM of Tris-HCl, 10 mMof MgCl₂ and 140 mM of KCl, pH 8.3 were prepared. The reaction solutionwas heated to 65° C. for 10 minutes and then cooled to room temperatureto allow annealing of the primer to the 3′ terminal of the sequencecoding for the variable region of mRNA.

[0114] After adding 2 μl of mouse Moloney virus reverse transcriptase(21 U/μl, Boehringer) to the reaction solution, the resulting solutionwas maintained at 42° C. for 1 hour to allow synthesis of the cDNA. Thesame strategy was used to clone the sequence coding for the variableregion of the light chain using VK1FOR as the primer.

[0115] B) Amplification of the DNAs Coding for the Variable Regions

[0116] For amplification of the sequence coding for the variable regionof the heavy chain, 10 μl of the mixture obtained in A) was added to 50pmoles of each of the primers (1 and 3), 250 μM of each of dATP, dTTP,dCTP and dGTP, 67 mM of Tris-HCl, 10 mM of MgCl₂, 17 mM of ammoniumsulphate, 200 μg/ml of gelatine and 4 units of Taq polymerase(Boehringer) then made up to 100 μl with water. The reaction solutionwas covered with a layer of viscous paraffin (liquid) and subjectedfirst to 3 amplification cycles with annealing at 45° C. and then to 30cycles where each cycle comprised 1 minute of denaturation of thenucleic acids at 95° C., 1 minute of annealing of the primers at 50° C.and 2 minutes of elongation at 72° C.

[0117] After amplification the reaction solution was extracted twicewith phenol-chloroform. The ds cDNA was then precipitated with ethanoland after separation by means of centrifugation was taken up in 100 μlof water and stored at 4° C.

[0118] The same procedure was used to amplify the sequence coding forthe variable region of the light chain using as primers VK₁FOR andVK₁BACK.

[0119] The cDNAs coding for said variable regions were sequenced by theSanger method (Sequenase kit version 2.0 USB) and the nucleotide andamino acid sequences are reported in FIGS. 1 and 2.

EXAMPLE 3

[0120] Construction of Recombinant Vectors

[0121] The DNAs (10 μl) derived from the amplification region weredigested with the restriction enzyme pairs PstI and BstEII, and KpnI andBglII and the digestion products were subsequently fractionated onpolyacrylamide gels (8%) by means of electrophoresis. Two bands werethen eluted containing respectively a DNA fragment of about 309 bpcomprising the gene VH5E8 delimited at the 5′ end by a PstI site and atthe 3′ end by a BstEII site and a DNA fragment of 312 bases comprisingthe gene VK5E8 delimited at the 5′ end by a KpnI site and at the 3′ endby a BglII site. The DNA fragments were eluted from the gel and clonedat the PstI and BstEII, and KpnI and BglII sites of the vector pSMA-pSMDobtained in examples 1 and 2.

[0122] By means of transformation and selection techniques of thepositive clones, the recombinant plasmids, namely pSM507, pSM438, pSM443and pSM442 were isolated, with characteristics as reported in table 1.

EXAMPLE 4

[0123] Expression and Secretion of scFv in B.subtilis

[0124] Cells of B.subtilis SMS300 were rendered competent as follows:the overnight culture in VY medium (Veal Infusion Broth 25 g/l, YeastExtract 5 g/l) was diluted 1:10 with minimum medium to which 5 mM MgSO4,0.5% glucose, 0.02% casamino acids and 50 μg/ml of essential amino acids(MMG1) were added, and grown for 4.5 hours at 37° C. and then furtherdiluted 1:5 with minimum medium to which of 5 mM MgSO4, 0.5% glucose, 5μ/ml of essential amino acids and 0/01% of casamino acids (MMG2) wereadded.

[0125] Aliquots (1 ml) of said dilution were transformed with 1 μg ofeach recombinant vector and incubated at 37° C. for 90 minutes withvigorous agitation.

[0126] After selection of the transformants on VY medium containingchloramphenicol, the single colonies were inoculated into 100 ml flaskscontaining 10 ml of VY (DIFCO) with 5 μ/ml of chloramphenicol and grownovernight at 37° C.

[0127] Six ml of each culture were then centrifuged at 15,000 rpm for 2minutes at 4° C. in order to separate the pellet of cells from theculture in which the exocellular proteins were isolated. In practice, 40μl of culture were added to 10 μl of loading buffer of the followingcomposition: 125 mM Tris-HCl pH6.8, 3% sodium dodecyl sulphate (SDS),20% glycerol, 3% beta-mercaptoethanol and 0.025% bromophenol blue, thenheat denatured (100° C.) and loaded onto polyacrylamide gel—SDS at 15%.

[0128] In parallel, the intracellular proteins were extracted afterhaving resuspended the pellet of cells in 460 μl of 50 mM buffercontaining 25% sucrose in TE pH 8.00. The mixture was incubated in thepresence of 12 μl of a 40 mg/ml lysozyme solution and 96 μl of EDTA 0.5M pH8, first at 37° C. for 30 minutes and then at 4° C. for 10 minutes.After adding 500 μl of 1% Triton^(R), 50 mM. of Tris-HCl pH 6.8 and 63mM EDTA cell lysis was completed by sonication for 2 minutes in ice.

[0129] Centrifugation at 6,500 rpm for 10 minutes at 4° C. separated thesupernatant (soluble fraction) from the pellet (insoluble fraction). Thetwo protein fractions were made up to a volume of 1.2 ml with loadingbuffer and were boiled for 5 minutes to obtain denatured and reducedproteins. Subsequently 10 μl of each protein preparation were analysedon polyacrylamide gel (15%) under denaturing and reducing conditions.

[0130] After electrophoresis at 40 mA for about 3 hours, the proteinbands on the two gels were observed by staining with Coomassie blue,transferred in parallel onto nitrocellulose filter (Schleicher andSchull 0.45 μm) and treated with rabbit serum producing anti-TAGantibodies and goat anti-rabbit IgG antibodies conjugated to peroxidase(Amersham). After staining with Coomassie blue, the following resultswere obtained: TABLE 1 Production of scFv5E8 in B. subtilis plasmidleader promoter total yield soluble secretion pSM507 LS (I) npr 30 mg/l100% pSM438 pelB npr 39 mg/l  25% pSM443 LS (I) Pc 10 mg/l 100% pSM442pelB Pc 200 mg/l  0

EXAMPLE 5

[0131] Characterisation of the Antibody

[0132] The antibody ScFv5E8 was isolated from the culture medium ofB.subtilis SMS300 (pSM507) cells by means of gel filtration on columnsTSK125+TSK250 with a flow of 1 ml/minute, using PBS as eluent and aninjection volume of 2 ml. Fractions were collected every 2 minutes at2.0 ml/fraction and each fraction was injected onto BIAcore^(R) toverify immunoreactivity with the antigen alpha hCG fixed to the sensor.

[0133] The results are reported in the following table. TABLE 2 scFv5E8MAb 5E8 Kass 3.952 × 10⁵ 1.415 × 10⁵ Kdiss 6.386 × 10⁻⁴ 2.300 × 10⁻⁶ KD1.616 × 10⁻⁹ 1.696 × 10⁻¹¹ KA 6.173 × 10⁸ 5.882 × 10¹⁰

[0134] where: Kass and Kdiss are the constants of kinetic associationand dissociation; KD is the dissociation constant at equilibrium; KA isthe affinity constant.

[0135] The results show that the association constant for scFv5E8 issimilar to that of MAb 5E8 while the Kdiss is about 300 times greaterthan that of the monoclonal antibody of origin. These values arecomparable with those obtained by Borrebaeck et al., (Biotech. 10:697-698, 1992) which compare a monoclonal antibody and itsmonofunctional derivative (Fab) prepared by means of proteolysis andindicate the maintenance of interaction characteristics at the bindingsite level of scFv produced in B.subtilis compared with the monoclonalantibody of origin.

1. An expression and secretion recombinant vector of B.subtilis whichcomprising: 1) the promoter of the gene coding for neutral protease inB.subtilis BGSC 1A341; 2) the secretion sequence (I) 5′ ATG AGA AGC AAAAAA ACG CGT ATC AGC    TTG TTG TTT GCG TTA ACG TTA ATC TTT    ACG ATGGCA TTC AGC GGC CGC TCT GCC    ATG GCC 3′; and

3) a DNA sequence coding for an antibody in single molecule form havingthe sequence VH/VK-L-VK/VH-(TAG)_(n where:) VH and VK are the variableregions of the heavy and light chains of an antibody of interest; L isthe peptide linker Val-Ser-Ser-(Gly₄-Ser)₃; TAG is a peptide recognisedby anti-peptide antibodies and n is 1 or
 0. 2. The recombinant vector asdefined in claim 1, wherein the peptide TAG has the amino acid sequenceMet-Glu-Glu-Leu-Met-Ile-Glu-Gly-Arg.
 3. The recombinant vector asdefined in claim 1, wherein VH and VK are the variable regions of theheavy and light chains of the monoclonal antibody MAb5E8 specific forsub-unit alpha of human chorionic gonadotropin.
 4. The recombinantvector as defined in claim 1, filed as ATCC
 69173. 5. The recombinantvector as defined in claim 1, obtained by: a) synthesizing anoligonucleotide comprising the secretion sequence (I), the sequencecoding for the peptide linker and, possibly, the sequence coding for thepeptide TAG, where said oligonucleotide contains unique restrictionsites allowing the insertion of the sequence coding for the variableregion of the heavy or light chain of the antibody downstream of thesecretion sequence (I) and upstream of the linkage peptide and theinsertion of the sequence coding for the variable region of the light orheavy chain of the antibody downstream of the peptide linker andupstream of the peptide TAG; b) cloning said oligonucleotide in aplasmid vector comprising the promoter of the gene for neutral proteasein B.subtilis BGSC 1A341; c) cloning the DNA sequences coding for thevariable region of the heavy and light chains of the antibody ofinterest in the restriction sites of the oligonucleotide; and finally d)isolating the recombinant vector.
 6. The recombinant vector as definedin claim 5, wherein the oligonucleotide of stage a) has the sequence: 5′GAA TTC TTA TGA GAA GCA AAA AAA CGC GTA TCA    GCT TGT TGT TTG CGT TAACGT TAA TCT TTA CGA    TGG CAT TCA GCG GCC GCT CTG CCA TGG CCG CAC   AGG TCC AAC TCC AGC CTA TGG TCA CCG TCT CCT    CAG GTG GCG GTG GCTCTG GCG GTG GTG GGT CGG    GTG GCG GCG GAT CTG ACA TTC AAG GTA CCC CCT   GAG ATC TCA TGG AAG AAC TTA TGA TCG AGG GTA    GGT AAG TCG ACA AGC TT3′.


7. The recombinant vector as defined in claim 5, wherein the plasmidvector of stage b) is pSM308 ATCC
 68047. 8. The recombinant vector asdefined in claim 5, wherein the DNA sequences of stage c) code for thevariable regions of the heavy and light chain of the monoclonal antibodyMAb 5E8 specific for sub-unit alpha of human chorionic gonadotropin. 9.A host microorganism transformed with a recombinant vector as defined inclaim 1, wherein said microorganism is selected from the B.subtilisgroup.
 10. The microorganism as defined in claim 6, which is B.subtilisSMS300 (pSM507) ATCC
 69173. 11. A procedure for the exocellularpreparation of antibodies in single molecule form from B.subtilis,comprising: a) cultivating in a suitable culture environment a strain ofB.subtilis transformed with a recombinant vector as defined in claim 1;and b) separating and purifying the antibody thus obtained from theculture medium.
 12. A procedure as defined in claim 11, whereinB.subtilis is B.subtilis SMS300 (pSM507) ATCC 69173 and the secretedantibody is scFv5E8 specific for sub-unit alpha of human chorionicgonadotropin.
 13. An antibody in single molecule form obtained by theprocedure defined in claim 7, for diagnostic and therapeutic use. 14.The antibody in single molecule form scFv5E8 for use as a reagent forthe determination of endocrine tumours and tumours of trophoblasticorigin characterised by comprising the regions VH and VK having theamino acid sequences reported in FIGS. 1 and
 2. 15. The secretionsequence (I) of in claim 1, namely: 5′ ATG AGA AGC AAA AAA ACG CGT ATCAGC TTG    TTG TTT GCG TTA ACG TTA ATC TTT ACG ATG    GCA TTC AGC GGCCGC TCT GCC ATG GCC 3′


16. The DNA sequence of claim 15 coding for the secretion peptide: MetArg Ser Lys Lys Thr Arg Ile Ser Leu Leu Phe Ala Leu Thr Leu Ile Phe ThrMet Ala Phe Ser Gly Arg Ser Ala Met Ala.